TWIST, a member of the basic Helix-Loop-Helix (bHLH) family of transcription factors, may be involved in the regulation of genes contributing to osteogenic cell proliferation and differentiation. In addition, recent results demonstrate a possible role for TWIST in chondrocyte development. These observations imply that TWIST and other bHLH factors may play a critical role in bone and cartilage development. The major questions to be answered by the specific aims in this proposal are as follows: (1) "What is the precise functional role of TWIST, a basic Helix-Loop-Helix transcription factor (e.g., suppressor/activator/??), upon regulating the expression of differentiation genes, during the early stages of bone and cartilage development?" and (2) "What is the biological significance of multiple TWIST transcripts in the regulation of early bone and cartilage development?" Four studies are proposed to answer these questions. The first study is designed to define the expression of TWIST transcripts in developing mouse, rat and human cell lines, under various in vitro conditions, where cells can be maintained in a proliferative or differentiated state, either as osteoblasts or chondrocytes. Second, in vivo expression studies using human, rat, and mouse primary bone and cartilage cell cultures, in addition to a variety of tissue sections, will be analyzed using TWIST antibodies by immunohistochemistry and by in situ hybridization (ISH) methods using RNA probes for several bone and cartilage specific markers, including TWIST. Observations gained from these two studies should point to transcriptional regulation patterns of the bone and cartilage marker genes in relation to TWIST, and help to determine the molecular mechanisms involved in early bone and cartilage formation. The third set of experiments are designed to study the mechanism of TWIST: bHLH heterodimerization and binding to the promoter regions of three specific early bone and cartilage marker genes (Alkaline phosphatase, Type I, and Type II Collagens). Results obtained from the in vitro binding studies and CAT transfection assays are expected to reveal a functional role for TWIST during the processes of both osteoblast and chondrocyte differentiation. The fourth study will determine the chromosomal location of TWIST on both human and mouse chromosomes. A clearer understanding of the function played by TWIST and other bHLH molecules to the development of normal bone and cartilage cells will contribute in the ability to monitor the effects bHLH factors may have on a variety of clinical diseases related to abnormal bone and cartilage development. Where correlations are found to exist, further studies will be performed to explore these molecular mechanisms which may contribute to possible future clinical applications.